Journal
EXPERIMENTAL CELL RESEARCH
Volume 322, Issue 2, Pages 227-235Publisher
ELSEVIER INC
DOI: 10.1016/j.yexcr.2014.01.002
Keywords
Astrin; Cell cycle; 25-Hydroxycholesterol; ORP8; Oxysterol-binding protein; SPAG5
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Funding
- NSFC, China [30971104]
- National Basic Research Program of China [2012CB517502]
- Fundamental Research Funds for the Central Universities, China [21610609]
- Sigrid Juselius Foundation
- Magnus Ehrnrooth Foundation
- Finnish Foundation for Cardiovascular Research
- Liv och Halsa Foundation
- Novo Nordisk Foundation
- Novo Nordisk Fonden [NNF11OC1014724] Funding Source: researchfish
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We earlier identified OSBP-related protein 8 (ORP8) as an endoplasmic reticulum/nuclear envelope oxysterol-binding protein implicated in cellular lipid homeostasis, migration, and organization of the microtubule cytoskeleton. Here, a yeast two-hybrid screen identified Homo sapiens sperm associated antigen 5 (SPAG5)/Astrin as interaction partner of ORP8. The putative interaction was further confirmed by pull-down and co-immunoprecipitation assays. ORP8 did not colocalize with kinetochore-associated SPAG5 in mitotic HepG2 or HuH7 cells, but over-expressed ORP8 was capable of recruiting SPAG5 onto endoplasmic reticulum membranes in interphase cells. In our experiments, 25-hydroxycholesterol (25OHC) retarded the HepG2 cell cycle, causing accumulation in G2/M phase; ORP8 overexpression resulted in the same phenotype. Importantly, ORP8 knock-down dramatically inhibited the oxysterol effect on HepG2 cell cycle, suggesting a mediating role of ORP8. Furthermore, knock-down of SPAG5 significantly reduced the effects of both ORP8 overexpression and 25OHC on the cell cycle, placing SPAG5 downstream of the two cell-cycle interfering factors. Taken together, the present results suggest that ORP8 may via SPAG5 mediate oxysterol interference of the HepG2 cell cycle. (C) 2014 Elsevier Inc. All rights reserved.
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